Use of the CRISPR/Cas9 technique to increase the ascorbic acid content in lettuce leaves

In this thesis, the CRISPR/Ca9 technique was used to knock out three negative regulators of the Ascorbic Acid (AsA) biosynthesis pathway, in order to have an increase of Ascorbate in Lactuca sativa cv. Cobham Green leaves. The three negative regulators on which we have decided to apply this technique are: CSN5B and CSN8, encoding two subunits of the protein complex named Photomorphogenic COP9 Signalosome (CSN) and Monodehydroascorbate reductase (MDHAR) gene. In particular, CSN5B and CSN8 proteins are involved in the ubiquitination GDP-D-Mannose pyrophosphorylase (GMP) enzyme in the dark and in its degradation through the 26S proteasome, leading to a decreased AsA content in Arabidopsis leaves. The MDHAR gene is involved in the recycling pathway of AsA and one of its isoforms has been shown to act as a negative regulator of ascorbate production in tomatoes. In lettuce genome, four different isoforms of MDHAR gene have been identified and we decided to knockout all the isoforms for a total of six target genes. Four gRNAs were selected for each target gene and assembled into a plasmid using the Golden Gate Cloning technique. Each plasmid was delivered into the plant via the Agrobacterium tumefaciens strain LBA4404, infecting 4-day-old lettuce cotyledons. 139 plants were obtained through the regeneration process, of which 122 were transformed. In transformed plants, gRNAs regions were amplified using specific primers to check the editing. From the sequencing it was found that fourteen plants had mutations in specific sites, in particular we obtained: two mutants from MDHAR1 plants, seven from MDHAR2 plants, one from MDHAR3 plants, three from MDHAR4 plants and one from CSN8 plants. No editing has been highlighted for the CSN5B gene. Most of the mutations led to frameshift alteration resulting from indels (base insertion and deletion). These types of mutations generated a stop codon in the coding sequence that could lead to truncated or no longer functional proteins, thus knocking out the gene sequence. Our results demonstrate that the engineered gRNA-Cas9 can accurately generate DSBs at specific sites in the plant genome, leading to targeted genetic mutations introduced by the NHEJ DNA repair machinery in lettuce.